Monoclonal antibodies (MAbs) facilitate biochemical analysis of proteins by its highly-specific recognition of a single epitope and its availability in large amounts. We are isolating MAbs to the proteins encoded by various human protooncogenes. Thus far, antibodies against the myc, ets-1 and ets-2 proteins have been produced and are being used to investigate their functions. This year six monoclonal antibodies were prepared from the mice immunized with a bacterially-expressed human ets-2 protein. These antibodies specifically recognize the two human ets-2-encoded proteins, p56 and p54, but failed to react with the proteins of chicken, mouse, rat, bovine, and monkey. Thus, they appear to recognize epitopes specific to human ets-2 protein. Immunoblot analysis with the bacterially-expressed human ets-2 protein, which had been partially digested by Staphylococcal V8 protease, indicated that at least 3 distinct epitopes in the B domain of the ets-2 protein are recognized by these antibodies. Immunoprecipitation experiments comparing native and denaturing conditions suggested that the domain of the ets-2 protein detected by the monoclonal antibodies is masked in its native condition either by protein folding or by interacting proteins. In further characterization of the human ets-2 protein enabled by the monoclonal antibodies, in vitro DNA binding activity associated with the protein was demonstrated. The MAb prepared previously against the human ets-1 protein has been used to purify the protein by affinity chromatography. All four major isoforms of the human ets-1 protein were thus purified and their N-terminal amino acid sequences were determined. This gave conclusive evidence for the identification of the heterogenous forms of the human ets-I protein.